Five member ring sulfenate esters and thiosulfinate esters

ABSTRACT

Disclosed are novel sulfenate esters and thiosulfinate esters that induce the expression of metabolic enzymes, particularly Phase II enzymes such as glutathione-s-transferase, DT-diaphorase and Ferritin H when administered to a subject. Also disclosed, such compounds are effective to displace a zinc ion from retroviral zinc finger nucleocapsid proteins effective to inhibit HIV replication. The present invention is further directed to novel methods of making these compounds, and any compounds produced by the process of making these novel compounds.

The government may have an interest in this patent pursuant to researchfinds that were supplied in the form of a grant from the NationalInstitutes of Health grant # R15GM51021.

FIELD OF THE INVENTION

The invention relates generally to novel sulfenate esters andthiosulfinate esters containing five membered rings, and to methods ofmaking these compounds. The invention also relates to the field oftherapeutic compositions for use as anti-cancer chemotherapeutic orchemoprotective agents, and as anti-HIV agents.

BACKGROUND OF THE INVENTION

Various types of cancers occur throughout the body, and affect largenumbers of people. It is postulated that many of these cancers arecaused by foreign substances, also referred to as xenobiotics. Onemethod of limiting the carcinogenic effect of various xenobiotics is bypromoting steps or increasing levels of substances in the metabolicpathway that allow carcinogens to be metabolized into neutral forms thatare easily excreted from the body.

A simplified description of how xenobiotics, compounds foreign to thebody, are metabolized is that procarcinogens are metabolized by Phase Ienzymes to either (i) electrophilic metabolites, which may cause DNAdamage leading to DNA repair mechanisms or to cancer, or to (ii)nonelectrophilic metabolites that are further metabolized by Phase IIenzymes to produce detoxification products. Several steps in thispathway potentially neutralize xenobiotics, and therefore could beconsidered as logical targets for chemoprevention of cancer. Forexample, the induction or inhibition of phase I enzymes might promoteneutralizing metabolic steps, as could the induction of phase IIenzymes. Alternatively, the promotion of DNA repair is a potentiallypromising means of preventing the carcinogenic effect of xenobioticsthat are metabolized through Phase I enzymes to produce electrophilicmetabolites which damage nucleic acid structures.

The prevention of the development of cancer by administering drugsprophylactically has been termed chemoprevention. Chemoprevention is anemerging concept that envisages the active prevention of malignantprocesses. Chemoprevention involves the “use of specific natural orsynthetic substances with the objective of reversing, suppressing orpreventing carcinogenic progression . . . ” (Singh and Lippman, 1998). Anumber of such chemopreventive agents are currently in development forundergoing clinical trials.

Chemopreventive agents can be conceptually classified as “blocking” or“suppressive” agents. Blocking agents prevent cancer-producing compoundsfrom reaching or reacting with their critical target sites; suppressiveagents prevent the evolution of the neoplastic process in cells alreadyaltered by carcinogens (see Singh and Lippman, 1998 for review). Thus,chemopreventive agents can be provided either to high risk groups or tothe population at large.

Oltipraz can be considered to have both blocking and suppressiveactivities, and is being investigated for chemopreventive activity in anumber of cancers, including those of the bladder, prostate, breast,skin, lung, colon, and liver (Wattenberg, 1997). A large scale clinicaltrial of the ability of oltipraz to prevent aflatoxin F1-relatedhepatocellular carcinoma is currently underway in the People's Republicof China. However, oltipraz has been associated with some toxicities,including photosensitivity/heat intolerance, GI effects, and neurologictoxicities (Dimitrov et al., 1992). Thus, the continued search forimproved chemopreventive agents is clearly warranted.

Phase II enzymes are responsible for the detoxification of reactiveelectrophilic and nucleophilic metabolites. Phase II enzymes includeNAD(PH):quinone oxidoreductase (facilitates metabolism of carcinogensthrough two electron reduction), glutathione S transferase (mediatesdeactivation of carcinogens through conjugation to reduced glutathione),manganese superoxide dismutase (reduces levels of superoxide anion)ferritin (reduces oxygen free radical formation by sequestering iron),and others (Talalay, 1989). Glutathione S-transferases (GST) are phaseII enzymes that catalyze the reaction of glutathione, a tripeptide, withelectrophiles such as epoxides, alkyl and aryl halides andα,β-unsaturated ketones. Glutathione conjugation serves to deactivateelectrophiles, therefore making them less toxic and carcinogenic andmore easily excreted by the body.

In the reaction between glutathione and an electrophile, the first stepinvolves the binding of glutathione (GSH) to theglutathione-S-transferase enzyme (GST). The enzyme is known to lower thepKa of the thiol from 9.0 to ˜6.5. The thiolate ion of glutathione thenreacts with electrophiles to produce the less toxic glutathioneconjugates.

Several sulfur-containing compounds are known to elevate levels of GSTin rats and mice. These include allicin (Talalay, et al.), a naturalproduct found in garlic, and Oltipraz 10, which is undergoing clinicaltrials at the time of the present disclosure.

Oltipraz and its derivatives (1,2-dithiole-3-thiones) are particularlyattractive because of two important characteristics. First,1,2-dithiole-3-thiones are monofunctional inducers. They only inducephase II enzymes and not phase I enzymes. Induction of phase I enzymescould enhance the production of activated carcinogens, thereforecomplicating any increased activity of phase II enzymes. Secondly,1,2-dithiole-3-thiones can induce higher levels of GSTs in severalorgans, providing possible protection against several types of cancer.

Because of this selective usefulness of sulfur-containing compounds thatdisplay an ability to induce high levels of GST in particular, industryis constantly seeking additional forms of such compounds, as well asreliable methods for their synthesis. As a result, several syntheseshave been reported for unsaturated five-membered cyclic sulfenate estersor γ-sultines (Bondarenko, et al.); however, none of the reportedsyntheses to date are very general. In 1970, King and co-workersreported that thermolysis of thiete 1,1-dioxide and 2-phenylthiete1,1-dioxide produced unsaturated sultines (King et al.) Thermolysis ofother substituted thiete 1,1-dioxides resulted in sulfur dioxideextrusion to yield various alkenes.

Braverman and co-workers have studied the electrophilic fragmentationand cyclization of allenic sulfones to unsaturated γ-sultines (Bravermanand Duar). Allenyl sulfone when reacted with bromine produced abromonium ion intermediate which cyclized to produce sultine. Thisreaction is unfavorably limited, however, because only brominesubstituted sultines can be synthesized.

Another synthesis of unsaturated sultines reported by Duboudin andco-workers required Grignard reagents formed from propargyl alcohols(Thomazeau et al.). The resulting Grignards reacted with sulfur dioxideby insertion into the carbon-metal bond. The sultines were obtained,however, in poor to moderate yields.

Accordingly, novel compounds that can induce GST and other enzymes ofthe Phase II response, and a method of reliably synthesizing suchcompounds in various forms while resulting in useful levels of thedesired product is still sought.

The tendency of HIV-1 to mutate to viral strains that are resistant toexisting therapeutic regimens is now well documented (Cohen, 1997). Thisresistance has fueled the search for antiviral drug targets that areconserved through mutations. The zinc finger containing nucleocapsidproteins of retroviruses would appear to be such a therapeutic targetsince they are conserved through mutations, are involved in early andlate phases of the viral replication cycle, and are chemically reactivetoward soft electrophilic reagents that can be prepared via rationalsynthetic structure-activity modification schemes (Rice and Turpin,1996). The structure of the HIV-1 nucleocapsid protein (NCp7) wasdetermined in the early 1990's (South et al., 1990; Chance, et al.,1992; Summers et al., 1992) and found to have theCys-Xaa₂-Cys-Xaa₄-His-Xaa₄-Cys zinc coordination sphere sequence withthe short tether links that are now sometimes referred to as a “zincknuckle”. At that same time Rice and co-workers first postulated thatthe Cys residues of zinc fingers could be chemically modified byelectrophilic attack. They first demonstrated the viability of thischemical postulate using 3-nitrosobenzamide (NOBA) (Rice et al., 1993).While NOBA reacted with the zinc fingers of NCp7 causing zinc ejection,it was reduced to an aromatic hydroxylamine. This class of compounds isknown to be quite mutagenic (Nohmi, et al., 1984) so the Rice groupinitiated a search for other soft electrophilic functional groups whichwould react with the sulfur rich zinc finger domain.

The disulfide functional group has the desired chemical reactivity andthe Rice group had access to a host of 2,2′-dithiobisbenzamides (DIBAs)(3) (submitted by Parke-Davis Pharmaceutical) via NCI's DevelopmentalTherapeutics Program (Rice et al., 1995; Rice et al., 1996; Tummino etal., 1996). These compounds were used by Rice et al. (1995 and 1996) andthe Parke-Davis group (Tummino et al., 1996) to definitively correlateability to eject zinc from the NCp7 protein with the ability of thecompounds to exert anti-HIV-1 activity.

Unfortunately, reaction of these acyclic disulfides (RSSR) (3) withintracellular reducing agents/nucleophiles such as reduced glutathioneproduced two thiols (RSH) which no longer react with the zinc fingers.This observation lead to a search for cyclic disulfides and analogsthereof, particularly for a molecule in which the 2 thiols could notdissociate from one another, thereby effectively rendering redox returnto the disulfide impossible. Lamar Field's group had deposited a numberof 1,2-dithiolanes (4) and 1,2-dithianes (5) in NCI's chemicalrepository and the Rice group screened a number of these (Rice et al.,1997).

Two of the six 1,2-dithiolanes (6, 7) and two (8, 9) of the 131,2-dithianes screened were active against HIV-1_(RF) replication inCEM-SS cells in the XTT-based cytoprotection assay, were active againstreplication of monocytotropic HIV-1_(ADA) in fresh humanmonocyte/macrophage cultures and promoted ejection of zinc from the NCp7protein. Both the XTT and monocyte/macrophage assays were used becauseinfection of monocyte/macrophage cultures is thought to resembleinfection of the nonproliferating pool of cells in vivo, while theparameters for the CEM-SS cells (XTT assay) are thought to reflect moreclosely the parameters for highly proliferating cells such as bonemarrow and intestinal cells. The CEM-SS cell (XTT) assay is a moresensitive indicator of toxicity and all compounds disclosed herein havebeen screened in this assay.

TABLE 1 Anti-HIV-1 and Zinc Ejection Screening of 6, 7, 8, 9

Monocyte/ XTT Macrophage Assay (μM) Assay (μM) # NSC # EC₅₀ IC₅₀ EC₅₀IC₅₀ TSQ NCp7 Assay 6 661127 34 200 N/A* N/A* 7 661126 9.8  30 21 >10018 8 624151 6.6 184 8.0 >100 17 9 624152 13 135 9.1 >100 20 N/A* Nomaterial was available for further testing.

The most active, least toxic compound, NSC 624151 (8), was then screenedfor of antiviral action against a variety of strains of HIV-1 and foundto be active (3-69 μM depending on strain) against all tested strains.NSC 624151 (8) was found to promote zinc ejection from the NCp7 proteinbut have no other observable effect on enzymatic activities of the viralreplication cycle.

The antiviral mechanism of action is postulated to be electrophilicattack of the oxidized organosulfur compounds on thenucleophilic/reducing cys residues present in the zinc fingers.Structurally related organosulfur compounds are known to be softelectrophiles and susceptible to nucleophilic attack by softnucleophiles such as thiols and thiolate anions (Talalay et al., 1988).These types of compounds are also substrates for glutathioneS-transferases, a family of enzymes involved in detoxification ofelectrophiles and chemoprevention of cancer (Wilce and Parker, 1994);Coles and Ketterer, 1990; Armstrong, 1991), and as discussed in thepreceding paragraphs. Oltipraz (10) has also been shown to prevent HIV-1replication (Prochaska et al., 1993). Since reduced glutathione is apotential competing intracellular nucleophile/reducing agent present inhigh concentrations in all cells, NSC 624151 (8) was also screened inthe XTT cytoprotection assay in the presence of a two fold excess ofglutathione (GSH). Antiviral activity was retained but the EC₅₀ didincrease by a factor of almost 10 from 6.6 μM to 64.2 μM. Toxicity wasalso decreased with GSH treatment with IC₅₀ going from 184 μM to >200μM. The competing nucleophile/reducing agent glutathione does reduce butdoes not eliminate the anti-HIV activity of these compounds.

After discovery that the cyclic dithianes and dithiolanes still maintainanti-HIV-1 activity even in the presence of reduced glutathione, theRice group began to tackle the critical question of zinc fingerspecificity. Can compounds be produced which will selectively targetretroviral nucleocapsid protein zinc fingers without affecting othercellular zinc fingers? This group has now shown that dithiane (8) doesnot affect poly (ADP-ribose) polymerase (PARP) activity or alterspecific binding of the zinc finger containing transcription factors Sp1and GATA-1 to their DNA targets (Huang et al., 1998). Lastly, they alsoexamined the effect of NSC 624151 (8) on in vitro transcription usingHeLa nuclear extract. A CMV promoter was used to drive the transcriptionof a duck HBV sequence and 8 did not significantly alter thistranscriptional process either.

Maynard and Covell have came out with a density functional theory (DFT)analysis, in combination with protein-ligand docking methods, forpredicting reactivity of NCp7 with electrophilic reagents (Maynard etal., 1998). They found that the molecular property of these softelectrophiles that correlated most strongly with their reactivity towardNCp7 and zinc ejecting ability was the ratio of electronegativity tohardness (χ²/η). This quantity is related to the capacity of anelectrophile to promote a soft, covalent bond forming reaction.Calculation of χ²/η for a variety of zinc ejectors was presented alongwith protein-ligand docking analysis.

From the DFT calculations, they observed a linear correlation betweenNCp7 reaction rates and the ligand's capacity to promote a soft,covalent reaction, χ²/η. The most reactive DIBA compounds (3,R′=C(O)NH-phenyl-SO₂NH₂ and R′=C(O)NH-phenyl-SO₂NHC(O)CH₃ had χ²/ηvalues of 0.2935 and 0.3098 respectively. The next most reactivecompound, dithiane, (9) had a χ²/η value of 0.2260. Compounds with χ²/ηvalues of less than 0.2 exhibited relatively poor zinc ejecting ability.

The NMR structure of NCp7 was then used as a fixed model for liganddocking analysis. Zinc finger 1 and zinc finger 2 docks were examinedand Fukui functions of the Cys thiolates (S¹⁵, S¹⁸, and S²⁸ in finger 1and S³⁹ and S⁴⁹ in finger 2) were calculated and their proximity to theligand electrophilic sites in the best scoring docks were calculated.Zinc finger 2 is known to be 7 fold more reactive (Rice et al., 1995;Rice et al., 1996; Tummino et al., 1996; Huang et al., 1998) than zincfinger 1 with the DIBA compounds (3) and this observation correlatedwell with calculated binding proximities. Frontier molecular orbitalCys⁴⁹ HOMO to electrophile LUMOs were then calculated and dithiane (9)showed high orbital overlap consistent with its chemical reactivity.This ligand docking analysis predicted a saddle shaped docking domain ofeach zinc finger and two nearly equivalent binding sites (designated αand β) for most small molecules inside the saddle.

Rice, Turpin and co-workers have reported new compounds capable ofbinding to both the α and β sites of zinc finger 2 and they included theCovell/Maynard calculations of their most likely binding conformations(Rice et al., 1999). In this study, they abandoned the disulfide linkagein 3 in favor of a thioester linkage (11). As mentioned earlier, acyclicdisulfides can be deactivated in this chemistry by reduction to thethiolates. The monomeric thiolates can then simply diffuse away from oneanother rather than recombine after reoxidation. Cyclic compounds suchas 9 do not suffer from this functional group disadvantage.

Extensive synthetic work reported in this paper resulted in theidentification of pyridinoalkanoylthiolesters (PATEs) (11) as compoundsthat could span both a and β docking sites and provide a softelectrophilic thioester group in close proximity to Cys⁴⁹. Thesecompounds had antiviral EC₅₀'s in the XTT cytoprotection assay of 5-6 μMand exhibited zinc finger reactivity, as measured by the Trp37fluorescence decrease assay of 3-4 relative fluorescence units/min over30 minutes.

The inventor is also familiar with work in which 32 different sulfurheterocycles and sulfur containing transition metal complexes werescreened in the XTT cytoprotection assay and zinc ejection assaydescribed herein above. Of the 18 metal complexes screened, only 1 (31)had antiviral activity and it did not have an EC₅₀. Cytoprotection of39% was observed at 100 μM and its IC₅₀ was 154.5 μM.

It would be advantageous, therefore to provide specific antiviralagents, capable of ejecting zinc from viral zinc finger configurations,in order to effectively address the mutational defense mechanisms ofHIV. The present disclosure is believed to address this deficiency inthe art, by providing novel compounds with anti-HIV activity and thatare also useful in the prevention of certain cancers.

SUMMARY

The present invention provides novel sulfenate esters and five memberedring thiosulfinate esters that are shown herein to have usefulbiological activities. The disclosed compounds may be used intherapeutic formulations to be administered to human or animal subjectswho have been exposed to, or may be exposed to certain cancer causingxenobiotics, or to humans or animals subject to developing certain typesof cancer, or to humans infected with HIV. The disclosed compounds mayalso be administered with the goal of preventing cancer in normal orhealthy subjects.

The present disclosure includes compositions that containpharmaceutically active agents that are effective in the treatment orprevention of various neoplastic diseases that include, but are notlimited to cancer of the bladder, prostate, breast, skin, lung, colon,liver, or pancreas, including invasive and non-invasive adenocarcinomas,and other cancers that occur in a tissue that expresses GST's or relatedmetabolic enzymes, or in the treatment or management of a retroviralinfection such as an infection with the human immunodeficiency virus(HIV).

An effective dose would include daily dosages of from about 1, 2, 3, 5to about 125, 300, 500, 600, or even about 1000 mg/kg/day of the activeagents. Agents may be administered in a single daily dose, or split into2, 3 or more doses to be taken throughout the day. Preferred regimensmay include taking the compositions daily for a week or more, forseveral weeks, and even daily or weekly for a period of about 12 weeksor more. If used in chemoprevention, long term administration (years) isenvisioned. The dosage and administration will be determined by apractitioner and will be based on the condition and need of theindividual patient, of course.

The active agents described herein may also be administered incombination with other active agents, particularly oncology agents, oranti-viral agents including, but not limited to anti-inflammatory drugssuch as ibuprofen, aspirin, acetaminophen, anti cancer drugs such asvarious retinoids, including but not limited to 13-cis-retinoic acid,retinol, or 4-hydroxyphenylretinamide, other possible anti-cancer agentssuch as beta-carotene, piroxicam, oltipraz, difluoromethylomithine,glycyrrhetinic acid, N-acetylcysteine, sodium molybdate,alpha-angelicalactone, alpha-tocopherol, coumarin, ellagic acid,flavone, indole-3-carbinol, d-limonene, phenethylisothiocyanate,mechlorethamine, chlorambucil, cyclophosphamide, melphalan, ifosfamide,methotrexate, 6-mercaptopurine, 5-fluorouracil, cytarabine, gemcitabine,vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, etoposide,irinotecan, topotecan, doxorubicin, bleomycin, mitomycin, carmustine,lomustine, cisplatin, carboplatin, interferons (interferon A),asparaginase, zidovudine, didanosine, zalcitabine, stavudine,lamivudine, nevirapine, delavirdine, saquinavir, indinavir, ritonavir,and nelfinavir.

The present invention may be described, therefore in certain preferredembodiments as a composition comprising a five membered ringthiosulfinate ester, or a five-membered ring sulfenate ester, whereinthe five membered ring has structure (I)

wherein, when X is O or S;

R₁ is an alkyl, preferably with 1-6 carbon atoms and more preferablymethyl, phenyl, substituted phenyl, cyclohexenyl, or substitutedcyclohexenyl;

R₂ is methyl, phenyl or CO₂R₃, wherein R₃ is alkyl or phenyl; and

when X is O and R₁ is phenyl, R₂ is not methyl. As used herein, chemicalelement abbreviations are given their art-accepted meanings. Forexample, O indicates Oxygen, S indicates Sulfur, and C indicates Carbon,Me indicates a methyl group, Et indicates an ethyl group, Pr indicates apropyl group, iPr indicates an isopropyl group, t-Bu indicates atert-butyl group, etc. In contrast, X, Y, and R are indicative ofvariables.

In preferred embodiments, when R₁ is a substituted phenyl, the phenyl issubstituted at the 4 position with a methoxy, t-butyl, alkyl ketone,sulfonamide, or trifluoromethyl group. Furthermore, when R₁ is asubstituted cyclohexenyl, the cyclohexenyl is preferably substituted inthe 2 position with a halogen, an alkyl, or a phenyl. In certainpreferred embodiments, X is O, R₁ is (4-MeO)phenyl, (4-t-Bu)phenyl orcyclohexenyl and R₂ is methyl. In certain preferred embodiments, X is O,R₁ is phenyl, and R₂ is alkyl, and preferably R₂ is methyl, ethyl,propyl, or isopropyl.

In certain embodiments, when X is O, R₁ is phenyl, and R₂ is CO₂R₃,where R₃ is preferably methyl, ethyl, propyl or isopropyl.Alternatively, when X is O, R₁ is cyclohexenyl, and R₂ is alkyl,preferably methyl, or phenyl. Furthermore, in certain embodiments, whenX is O, R₁ is (4-MeO)phenyl and R₂ is CO₂R₃, or when X is O, R₁ may be(4-trifluoromethyl)phenyl, 4-acylphenyl, 4-sulfonamidylphenyl, or acyclohexenyl with a halogen, preferably chloride, fluoride, bromide oriodide substituted at the 2 position. In certain embodiments, when X isO, R₁ is a cyclohexenyl with an alkyl substitution at the 2 position,and preferably an alkyl comprising 1 to 4 carbon atoms, or R₁ is acyclohexenyl with a phenyl substitution at the 2 position.

In certain preferred embodiments of the compositions described above,when X is S, R₁ is phenyl or an alkyl with 1-6 carbon atoms, preferablymethyl, and R₂ is CO₂R₃, where R₃ is preferably methyl, ethyl,isopropyl, or propyl. In alternative embodiments, X is S, R₁ is(4-MeO)phenyl, (4-t-Bu)phenyl or cyclohexenyl, and R₂ is methyl. Instill further preferred embodiments, X is S, R₁ is phenyl, and R₂ isalkyl, and preferably R₂ is methyl, ethyl, propyl, or isopropyl. Incertain embodiments, X is S, R₁ is cyclohexenyl, and R₂ is alkyl,preferably methyl, or R₂ may be phenyl.

In further embodiments of the disclosed formulations, when X is S, andR₁ may be methyl, (4-trifluoromethyl)phenyl, 4-acylphenyl,4-sulfonamidylphenyl, or alternatively, R₁ is (4-MeO)phenyl and R₂ isCO₂R₃.

In certain embodiments, X is S, and R₁ is a cyclohexenyl with a halogensubstitution, preferably chloride, fluoride, bromide or iodide at the 2position, or alternatively R₁ is a cyclohexenyl with an alkylsubstitution at the 2 position, preferably an alkyl with 1 to 4 carbonatoms, or R₁ is a cyclohexenyl with a phenyl substitution at the 2position.

In certain embodiments, a composition as described herein will have anadditional element Y as shown in structure (II):

As can be seen, if the Y group is removed from structure II, the resultis structure I. In certain preferred embodiments, Y is O, R₁ is phenyland R₂ is methyl, phenyl or hydrogen. It is understood that any of thecompositions described herein as derived from structure I or II may becontained in a pharmaceutically acceptable carrier, and preferably apharmaceutical carrier, or pharmacological formulation prepared for oraladministration.

The present invention may also be described, in certain preferredembodiments as a pharmaceutical composition comprising a five memberedring thiosulfinate ester, or a five-membered ring sulfenate ester,wherein the five membered ring has structure (II) in an amount effectiveto induce glutathione-S-transferase (GST), DT-diaphorase (NQO1), orFerritin H expression when administered to a subject; and furtherwherein when X is O or S;

R₁ is an alkyl with 1-6 carbon atoms, preferably methyl, phenyl,substituted phenyl, cyclohexenyl, or substituted cyclohexenyl;

R₂ is methyl, phenyl or CO₂R₃, wherein R₃ is alkyl or phenyl; and Y isnot present or Y is O. It will be clear to those of skill in the art inlight of the disclosure, that when Y is not present, then structure (II)is equivalent to structure (I).

An embodiment of the disclosure may also be described as a compositionincluding a unit dose effective to inhibit or decrease the incidence ofcancer in a subject, such as an animal or human subject, and in certaincases, an amount effective to induce glutathione-S-transferase (GST),DT-diaphorase (NQO1), or Ferritin H or other phase II enzymes expressionin a subject when the composition is administered to the subjectperiodically. In certain embodiments, a subject to receive thiscomposition is a human or animal that has been exposed to, or issusceptible to being exposed to a carcenogenic xenobiotic agent. Incertain embodiments, a subject to receive this composition is anunaffected (normal) human. In certain embodiments, the describedcompositions may also be used to inhibit replication of certain viruses,including retroviruses, and in particular HIV. As such, the presentinvention includes pharmaceutical compositions in an amount effective toinhibit HIV replication when the composition is administered to asubject, and in particular to a human HIV patient.

As described herein an effective amount is from about 1 to about 1000 mgper dose, or from about 5 to about 600 mg, or even from about 100 toabout 500 mg. As a chemopreventive, for such dosages are preferably asingle daily dose taken orally, and may be administered for days, weeks,or even years. For treatment of HIV, such doses may be administeredperiodically in order to achieve continued inhibition of viralreplication. It is understood that these compositions may be used asadjunct therapy with any compatible chemotherapeutic agent in thetreatment of HIV, or in prevention of cancer.

Any numerical values recited herein include all values from the lowervalue to the upper value in increments of one unit provided that thereis a separation of at least two units between any lower value and anyhigher value. As an example, if it is stated that the concentration of acomponent or value of a process variable such as, for example,osmolality, temperature, pressure, time and the like, is, for example,from 1 to 90, preferably from 20 to 80, more preferably from 30 to 70,it is intended that values such as 15 to 85, 22 to 68, 43 to 51, 30 to32 etc. are expressly enumerated in this specification. For values whichare less than one, one unit is considered to be 0.0001, 0.001, 0.01 or0.1 as appropriate. These are only examples of what is specificallyintended and all possible combinations of numerical values between thelowest value and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 depicts data showing the induction of GST encoding mRNA in cellculture upon contact with compound 123.

FIG. 2 depicts data showing expression of a GST enzyme in cell cultureupon contact with compound 123.

DETAILED DESCRIPTION

The present invention provides novel and useful compositions thatinclude unsaturated five member ring containing sulfenate esters andthiosulfinate esters. These compositions are shown herein to have atleast one of the following useful biological activities, either asagents for the prevention or protection against certain cancers,especially activity as inducers of glutathione-S-transferase expression,NQO1 DT-diaphorase expression, Ferritin H expression, or expression ofother enzymes of the Phase II response, an activity useful in thedefense against cancer causing xenobiotic agents and cancer prevention,or activity as anti-HIV agents that eject the Zn²⁺ ion from a zincfinger nucleocapsid protein in HIV. Specific examples of the sulfenateesters provided include the novel GST-inducers4-methyl-5-(4-methoxy)phenyl-1,2-oxathiol-4-en-1-yl oxide and4-methyl-5-cyclohexenyl-1,2-oxathiol-4-en-1-yl oxide.

Additionally, the present disclosure provides methods of manufacturingthese and additional compounds that may be screened for their capacityto induce elevated levels of GST, NQ01 or Ferritin H, and other Phase IIenzymes, and may prove useful as cancer chemoprotectants or cancerchemotherapeutics. In general, the novel process of synthesizingcompounds disclosed herein includes the steps of making an alkynylalcohol; converting the alkynyl alcohol to an alkynyl tosylate; formingan iron alkynyl complex from an alkynyl tosylate; performingtransition-metal mediated [3+2] cycloaddition of the iron alkynylcomplex with sulfur dioxide or disulfur monoxide to produce an ironsulfenate ester or thiosulfinate ester; and from this, producing usefulsulfenate esters and thiosulfinate esters.

Pharmacological Compositions

The active compounds may be orally administered, for example, with aninert diluent or with an assimilable edible carrier, or they may beenclosed in hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic or prophylactic administration,the active compounds may be incorporated with excipients and used in theform of ingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Preparation of Sulfenate Esters

The following example describes in detail the preparation of two novelfive member ring sulfenate esters, compounds 121 and 123, which havebeen designated by the inventor as compounds ALH-556 (121) and ALH-562(123).

A. Synthesis of Alkynyl Alcohols 95 and 99-101

First, the appropriate alkynyl alcohols are synthesized. To produce theparticular compositions disclosed herein, 1-Ethynylcyclohexene, forexample, is deprotonated with butyl lithium and then condensed withparaformaldehyde (Brandsma and Verkruijsse). By this process,3-Cyclohexenyl-2-propyn-1-ol was produced in 82% yield (Baudouy, etal.). This compound had been reported previously without completespectroscopic data (Baudouy). 1-Ethynylcyclohexene (3.00 g, 28.6 mmol)was dissolved in diethyl ether (125 mL) and cooled to −78° C. 2.5 Mn-butyllithium in hexane (12.4 mL, 31.1 mmol) was added dropwise. Thesolution was allowed to stir at −78° C. for 1.5 h and thenparaformaldehyde (1.70 g, 56.5 mmol) was added. The reaction mixture wasallowed to warm to 25° C. overnight. The mixture was poured into sat.NH₄Cl solution (125 mL) and then extracted with diethyl ether (3×50 mL).The ether extracts were dried with MgSO₄ and the solvent was removedunder reduced pressure. The crude product was purified by columnchromatography on silica gel using 1:1 diethyl ether/pentane to yield alight yellow oil (3.17g, 23.3 mmol, 82.4%). ¹H NMR (CDCl₃): 6.09 (m, 1H), 4.35 (s, 2 H), 2.07 (m, 4 H), 1.62-1.57 (m, 5 H). EI HRMS (m/e)called for (M+)(C₉H₁₂O) 136.0888, found 136.0894.

General Procedure for the Synthesis of Substituted Phenyl PropargylAlcohols

Other useful alkynyl alcohols may be synthesized by the coupling of anappropriate aryl iodide with propargyl alcohol using Pd(PPh₃)₂Cl₂ andcopper (I) iodide (Yang and Burton; Kondo, et al.). For example,4-Iodoanisole was converted to 3-(4-Methoxy)-phenyl-2-propyn-1-ol in 98%yield (Wadsworth et al.). Alternatively,3-(4-tert-butyl)-phenyl-2propyn-1-ol was isolated in 85% yield from thecoupling of 1-tert-butyl-4-iodobenzene with propargyl alcohol. By way offurther example, the coupling reaction of 1-Iodo-nitrobenzene andpropargyl alcohol produced 3-(4-Nitro)-phenyl-2-propyn-1-ol in 89% yield(Harris, et al.).

The appropriate iodobenzene was dissolved in triethylamine (10-50 mL)and propargyl alcohol was added. The reaction mixture was thoroughlydeoxygenated using nitrogen. Pd(PPh₃)₂Cl₂ (0-01 mol %) and CuI (0.02 mol%) were added and the resulting solution was allowed to stir for 2 h at25° C. H₂O (20-40 mL) and ethyl acetate (20-40 mL) were added. Theaqueous layer was extracted with ethyl acetate (2×20 mL). The organicextracts were combined and dried with MgSO₄. The solvent was removedunder reduced pressure. The crude products were purified byrecrystallization or column chromatography on silica gel.

3-(4-Methoxy)-phenyl-2-propyn-1-ol.

4-Iodoanisole (5.00 g, 21.4 mmol), propargyl alcohol (1.25 mL, 21.4mmol), Pd(PPh₃)₂Cl₂ (0.150 g, 0.214 mmol) and CuI (0.082 g, 0.428 mmol)were reacted using the above procedure to yield a crude product whichwas purified by column chromatography using silica gel with 100% diethylether. The resulting off-white solid (3.40 g, 21.0 mmol, 98.3 %) provedidentical by spectroscopic comparison to previously reported material(Wadsworth, et al.).

3-(4-tert-butyl)-phenyl-2-propyn-1-ol.

1-tert-Butyl-4-iodobenzene (1.00 g, 3.84 mmol), propargyl alcohol (0.223mL, 3.84 mmol), Pd(PPh₃)₂CI₂ (0.027 g, 0.038 mmol) and CuI (0.0 15 g,0.077 mmol) were reacted using the above procedure to yield a crudeproduct which was purified by column chromatography using silica gelwith 100% diethyl ether. The resulting product was a light yellow solid(0.615 g, 3.27 mmol, 85.1%), m.p. 94-95° C. ¹H NMR (CDCl₃): 7.33 (m, 4H), 4.47 (s, 2 H), 1.90 (br s, 1 H), 1.29 (s, 9 H). Anal. Calcd forC₁₃H₁₆O: C, 82.94; H, 8.57. Found: C, 82.18; H, 8.54. E1HRMS (m/e) calcdfor M⁺ (Cl₁₃H₁₆O), 188.1201, found 188.1203.

3-(4-Nitro)-phenyl-2-propyn-1-ol.

1-Iodo-4-nitrobenzene (5.00 g, 20.1 mmol), propargyl alcohol (1.17 ml,20.1 mmol), Pd(PPh₃)₂CI₂ (0.140 g, 0.200 mmol), and CuI (0.076 g, 0.400mmol) were reacted using the above procedure to yield a crude productwhich was purified by recrystallization from ethyl acetate and pentane.The tan solid (3.18 g, 17.9 mmol, 89.3%) proved identical byspectroscopic comparison to previously reported material (Harris, etal.)

B. Preparation of Alkynyl Tosylates

The alkynyl alcohols produced from step one, may then be converted intoalkynyl tosylates. Propargyl alcohol was dissolved in diethyl ether (100mL) and p-toluene sulfonyl chloride (0.95 equiv.) was added. Thesolution was cooled to −15° C. and powdered potassium hydroxide (5.0equiv.) was added 1 equivalent at a time over 30-45 min. The reactionmixture was then allowed to stir at −15° C. for 90 min. Ice water (100mL) was then added and the mixture was extracted with diethyl ether(3×50 mL). The solution was dried with MgSO₄ and the solvent was removedunder reduced pressure. The product was then triturated with petroleumether (15 mL), cooled to −78° C., and the solvent was decanted. Theremaining product was dried under vacuum.

Tosylates 1-tosyl-3-cyclohexenyl-2propyne,1-Tosyl-3-(4-methoxy)phenyl-2-propyne, and1-Tosyl-3-(4-tert-butyl)phenyl-2-propyne were prepared in 92%, 84% and68% yield respectively. Tosylate 1-Tosyl-3-(4-nitro)phenyl-2-propyne wasprepared in 59% yield from 3-(4-Nitro)-phenyl-2-propyn-1-ol at 25° C.rather than the standard temperature of −10° C.

1-Tosyl-3-cyclohexenyl-2-propyne.

3-cyclohexenyl-2-propyn-1-ol (95)(3.04 g, 22.3 mmol), p-toluene sulfonylchloride (4.04 g, 21.2 mmol) and potassium hydroxide (6.26 g, 112 mmol)were reacted using the above procedure to yield the product (5.66 g,19.5 mmol, 91.8%) as a light yellow oil. ¹H NMR (CDCl₃): 7.79 (d, J=8.1Hz, 2 H), 7.31 (d, J=8.1 Hz, 2 H), 5.97 (m, 1 H), 4.81 (s, 2 H), 2.42(s, 3 H), 2.02 (m, 2 H), 1.92 (m, 2 H), 1.53 (m, 4 H). FAB LRMS (m/e):calcd. for (MH^(+)(C) ₁₆H₁₉O₃S): 29 1, found 29 1.

1-Tosyl-3-(4-methoxy)phenyl-2-propyne.

3-(4-methoxy)phenyl-2-propyn-1-ol (3.56 g, 22.0 mmol),p-toluene sulfonylchloride (3.97 g, 20.9 mmol), and potassium hydroxide (6.16 g, 110 mmol)were reacted using the above procedure to yield the product (5.50 g,17.4 mmol, 83.7%) as a white solid: m.p. 44-45° C. ¹H NMR (CDCl₃): 7.83(d, J=8.2 Hz, 2 H), 7.29 (d, J=8.2 Hz, 2 H), 7.18 (d, J=8.7 Hz, 2 H),6.78 (d, J=8.7 Hz, 2 H), 4.91 (s, 2 H), 3.78 (s, 3 H), 2.38 (s, 3H). FABLRMS (m/e): calcd. for (MH+)(Cl₁₇H₁₇O₄S): 317, found 317.

1-Tosyl-3-(4-tert-butyl)phenyl-2-propyne.

3-(4-tert-butyl)phenyl-2-propyn-1-ol (0.550 g, 2.92 mmol), p-toluenesulfonyl chloride (0.529 g, 2.77 mmol) and potassium hydroxide (0.819 g,14.6 mmol) were reacted using the above procedure to yield the product(0.649 g, 1.90 mmol, 68.4%) as a white solid: m.p. 83-84° C. ¹H NMR(CDCl₃): 7.83 (d, J=8.1 Hz, 2 H), 7.31-7.26 (m, 4 H), 7.16 (d, J=7.9 Hz,2 H), 4.93 (s, 2 H), 2.37 (s, 3 H), 1.28 (s, 9 H). Anal. Calcd forC₂₀H₂₂O₃S: C, 70.15; H, 6.48. Found: C, 70.09; H, 6.59.

1-Tosyl-3-(4-nitro)phenyl-2-propyne.

3-(4-nitro)phenyl-2-propyn- 1-ol (3.18 g, 18.0 mmol), p-toluene sulfonylchloride (3.25 g, 17.1 mmol), and potassium hydroxide (5.04 g, 89.8mmol) were reacted using the above procedure except that the reactionmixture was allowed to stir overnight at 25° C. to yield a yellow solid(3.32 g, 10.0 mmol, 58.8%): m.p. 103-104° C. ¹H NMR (CDCl₃): 8.14 (d,J=8.5 Hz, 2 H), 7.84 (d, J=8.0 Hz, 2 H), 7.40 (d, J=8.5 Hz, 2 H), 7.33(d, J=8.0 Hz, 2 H), 4.94 (s, 2 H), 2.40 (s, 3 H). Anal. Calcd. forC₁₆H₁₃NO₅S: C, 57.99; H, 3.95. Found: C, 57.7 1; H, 4.06.

C. Synthesis of Iron Alkynyl Complexes

The alkynyl tosylates obtained from step two are then reacted with thecyclopentadienyl iron dicarbonyl anion to yield desired iron alkynylcomplexes.

The iron anion was generated by stirring a THF solution of [CpFe(CO)₂]₂over a 1% sodium amalgam for 5 h. The anion was then added using adouble ended needle to a THF solution of the appropriate alkynyltosylate cooled to 0° C. The resulting mixture was allowed to warm to25° C. over 1 h. The solvent was removed by rotary evaporation. Theremaining residue was washed with pentane until the washes werecolorless. The pentane was removed by rotary evaporation. The crudeproduct was vacuum dried and purified by column chromatography onalumina.

The complex cyclopentadienyl (3-cyclohexenyl-2propynyl)dicarbonyl ironwas prepared in 84% yield from 1-Tosyl-3-cyclohexenyl-2-propyne.Likewise, iron alkynyl complexes cyclopentadienyl(3-(4-methoxy)phenyl-2-propynyl)dicarbonyl iron andcyclopentadienyl(3-(4-tert-butyl)phenyl-2-propynyl)dicarbonyl iron maybe prepared from tosylates 1-Tosyl-3-(4-methoxy)phenyl-2-propyne and1-Tosyl-3-(4-tert-butyl)phenyl-2-propyne in 72% and 97% yieldrespectively.

Cyclopentadienyl(3-cyclohexenyl-2-propynyl)dicarbonyliron.

The iron anion was generated from [CpFe(CO)₂]₂ (3.68 g, 10.4 mmol) andwas added to a THF solution of 1-Tosyl-3-cyclohexenyl-2-propyne (5.50 g,18.9 mmol) using the procedure outlined previously. The product wasobtained as a brown solid (4.68 g, 15.8 mmol, 83.5%): m.p. 52-53° C. IR(NaCl): 2932, 2002, 1944 cm⁻¹. ¹H NMR (C₆D₆): 6.10 (m, 1 H), 4.08 (s, 5H), 2.25 (m, 2 H), 1.89 (br s, 4 H), 1.41 (m, 4 H). ¹³C NMR (C₆D₆):216.88, 130.55, 123.22, 98.51, 85.98, 53.61, 30.47, 25.88, 22.88, 22.08,−18.19. Anal. Calcd for C₁₆H₁₆FeO₂: C, 64.89; H, 5.45. Found: C, 64.18;H, 5.45.

Cyclopentadienyl(3-(4-methoxy)phenyl-2-propynyl)dicarbonyliron.

The iron anion was generated from [CpFe(CO)₂]₂ (3.26 g, 9.23 mmol) andwas added to a THF solution of 1-Tosyl-3-(4-methoxy)phenyl-2-propyne(5.31 g, 16.8 mmol) using the procedure outlined previously. The productwas obtained as a dark red solid (3.87 g, 12.0 mmol, 71.5%): m.p. 60-61°C. IR (NaCl): 2003, 1949, 1505, 1238, 827 cm⁻¹. ¹H NMR (C₆D₆): 7.45 (d,J=8.0 Hz, 2 H), 6.66 (d, J=8.2 Hz, 2 H), 4.09 (s, 5 H), 3.21 (s, 3 H),1.95 (s, 2 H). ¹³C NMR (C₆D₆): 216-88, 158.93, 132.63, 118.96, 114.30,99.94, 85.99, 83.65, 54.70, −18.43. Anal. Calcd for C₁₇H₁₄FeO₃: C,63.38; H, 4.38. Found: C, 63.18; H, 4.66.

Cyclopentadienyl(3-(4-tert-butyl)phenyl-2-propynyl)dicarbonyliron.

The iron anion was generated from [CpFe(CO)₂]₂ (0.355 g, 1.01 mmol) andwas added to a THF solution of 1-Tosyl-3-(4-tert-butyl)phenyl-2-propyne(0.626 g, 1.83 mmol) using the procedure outlined previously. Theproduct was obtained as a dark red solid (0.615 g, 1.77 mmol, 97%): m.p.58-59° C. IR (NaCl): 2961, 2009, 1951, 825 cm⁻¹. ¹H NMR (C₆D₆): 7.53 (d,J=8.4 Hz, 2 H), 7.16 (d, J=8.4 Hz, 2 H), 4.08 (s, 5 H), 1.93 (s, 2 H),1.16 (s, 9 H). ¹³C NMR (C₆D₆): 216.78, 149.50, 131.11, 125.54, 123.80,101.06, 85.94, 53.95, 34.52, 31.22, −18.55. Anal. Calcd for C₂₀H₂₀FeO₂:C, 68.98; H, 5.79. Found: C, 69.22; H, 5.99.

D. Cycloaddition of Iron Alkynyl Complexes with Sulfur Dioxide

The transition-metal mediated [3+2] cycloadditions of the iron alkynylcomplexes with sulfur dioxide were performed as follows: The appropriateiron allenyl or propargyl complex was dissolved in CH₂Cl₂ (10-15 mL),purged with nitrogen and cooled to −78° C. Sulfur dioxide (10 mL) wascondensed at −78° C. into the iron complex solution. The reactionmixture was allowed to warm to 25° C. under nitrogen to allowevaporation of excess sulfur dioxide. The solvent was removed by rotaryevaporation and the remaining solid was vacuum dried.

Alkynyl complex cyclopentadienyl(3-cyclohexenyl-2propynyl)dicarbonyliron cyclized with SO₂ to producethe iron sulfenate estercyclopentadienyl(1-oxo-5-cyclohexenyl-1,2,oxathiol-4-3n-4-yl)dicarbonylironin 76% yield. Iron complexesCyclopentadienyl(3-(4-methoxy)phenyl-2-propynyl)dicarbonyliron andCyclopentadienyl(3-(4-tert-butyl)phenyl-2-propynyl)dicarbonyliron alsocyclized with SO₂ yielding the iron sulfenate esterscyclopentadienyl(1-oxo-5-(4-methoxy)phenyl-1,2-oxathiol-4-en-4-yl)dicarbonylironandcyclopentadienyl(1-oxo-5-(4-tert-butyl)phenyl-1,2-oxathiol-4-en-4-yl)dicarbonylironin 67% and 66% yield respectively.

Cyclopentadienyl(1-oxo-5-cyclohexenyl-1,2.oxathiol-4-en-4-yl)dicarbonyliron(117).

The iron alkynyl complex,Cyclopentadienyl(3-cyclohexenyl-2-propynyl)dicarbonyliron (1.50 g, 5.07mmol) was treated with SO₂ to generate the crude product, which waspurified by recrystallization from CH₂Cl₂/pentane to yield a yellowsolid (1.39 g, 3.86 mmol, 76.4%): m.p. 104-105° C. (dec.). IR (NaCl):2925, 2107,1966, 1101, 876 cm⁻¹. ¹H NMR (CDCl₃): 5.80 (t, J=1.1 Hz, IH), 5.35 (d, J=14.6 Hz, I H), 5.00 (d, J=14.6 Hz, I H), 4.93 (s, 5 H),2.16 (m, 4 H), 1.66 (m, 4 H). ¹³C NMR (CDCl₃): 213.63, 213.38, 153.11,148.81, 131.33, 131.07, 92.60, 85.35, 29.82, 25.62, 22.60, 21.63. Anal.Calcd for C₁₆H₁₆FeO₄S: C, 53.35; H, 4.48; Found: C, 52.67; H, 4.48. FABHRMS (m/e): calcd for (MH+)(C₁₆H₁₇O₄FeS), 361.0197; found 361.0195.

Cyclopentadienyl(1-oxo-5-(4-methoxy)phenyl-1,2-oxathiol-4-en-4-yl)dicarbonyliron(118).

The iron alkynyl complex,Cyclopentadienyl(3-(4-methoxy)phenyl-2-propynyl)dicarbonyliron (1.50 g,4.66 mmol) was treated with SO₂ to generate the crude product, which waspurified by recrystallization from CH₂Cl₂/pentane to yield a red-brownsolid (1.21 g, 3.13 mmol, 67.2%): m.p. 105-106° C. (dec.). IR (NaCl):2024, 1966, 1494,1242, 1099, 895 cm⁻¹. ¹H NMR (CDCl₃): 7.31 (d, J=8.1Hz, 2 H), 6.94 (d, J=8.1 Hz, 2 H), 5.51 (d, J=14.7 Hz, 1 H), 5.13 (d,J=14.7 Hz, 1 H), 4.75 (s, 5 H), 3.83 (s, 3 H). ¹³C NMR (CDCl₃): 213.50,213.30, 159.60, 152.89, 150.31, 131.64, 125.84, 114.02, 92.77, 85.50,55.30. Anal. Calcd for C₁₇H₁₄FeO₅S: C, 52-87; H, 3.65. Found: C, 52.69;H, 3.78.

Cyclopentadienyl(1-oxo-5-(4-tert-butyl)phenyl-1,2-oxathiol-4-en-4-yl)dicarbonyliron(119).

The iron alkynyl complex,Cyclopentadienyl(3-(4-tert-butyl)phenyl-2-propynyl)dicarbonyliron (0.615g, 1.77 mmol) was treated with SO₂ to generate the crude product, whichwas purified by recrystallization from CH₂Cl₂/pentane to yield a yellowsolid (0.484 g, 1.17 mmol, 66.5%): m.p. 59-60° C. IR (NaCl): 2961, 2023,1973, 1109, 897 cm⁻¹. ¹H NMR (CDCl₃): 7.42 (d, J=8.3 Hz, 2 H), 7.32 (d,J=8.0 Hz, 2 H), 5.52 (d, J=14.8 Hz, 1 H), 5.14 (d, J=14.8 Hz, 1 H), 4.73(s, 5 H), 1.33 (s, 9 H). ¹³C NMR (CDCl₃): 213.41, 213.29, 152.34,151.34, 150.59, 130.51, 130.02, 125.47, 92.88, 85.54, 34.70, 31.33.Anal. Calcd for C₂₀H₂₀FeO₄S: C, 58.27; H, 4.89. Found: C, 58.54; H,4.98.

E. Synthesis of Sulfenate Esters

Iron sulfenate ester of step four is then treated to produce the desiredend product: a sulfenate ester. One way to achieve such a resultincludes treating the iron sulfenate ester with perchloric acid toreplace the iron-ligand set with a proton. A complicated mixture ofproducts is produced from this reaction, however, none of which resemblethe desired product (120). Additionally, when sulfenate ester 117 wastreated with Stryker's reagent, known to deliver hydride by Michaeladdition, no reaction was observed (Mahoney, et al.).

Rather, a surprising and useful way of deriving the disclosedGST-inducing sulfenate esters from the precursor iron sulfenate esterswas developed. In a flame dried flask, CuI (3 equiv.) was suspended infreshly distilled THF (5-10 mL) and the solution was thoroughly purgedwith nitrogen and cooled to −100° C. Methyllithium (1.5 M in Et₂O, 6equiv.) was added slowly. The resulting solution was allowed to stir for30 min. at −100° C. In another flame dried flask, the appropriate ironcomplex was dissolved in THF (5-10 mL) and the solution was thoroughlypurged with nitrogen and cooled to −100° C. The iron complex solutionwas added to the cuprate solution using a double ended needle. Thereaction mixture was then allowed to stir at −100° C. for 2 h. Sat.NH₄Cl solution (10-20 mL) was then added. The aqueous layer wasextracted with diethyl ether (2×15 mL). The organic extracts werecombined and dried with MgSO₄. The solvent was removed by rotaryevaporation and the remaining oil or solid was vacuum dried. The crudeproduct was purified by column chromatography using silica gel at 0° C.with diethyl ether/pentane (1: 1).

4-Methyl-5-(4-methoxy)phenyl-1,2-oxathiol-4-en-1-yl Oxide (121).

Iron complex 118 (0.200 g, 0.518 mmol) was reacted with the cuprateformed from CuI (0.148 g, 0.777 mmol) and methyllithium (1.4 M in Et₂O,1.11 ml, 1.55 mmol) using the above procedure to produce the crudeproduct which was purified to yield an off-white solid (0.027 g, 0. 120mmol, 23 %): m.p. 74-75° C. IR (NaCl): 1602, 1501, 1246, 1116 cm⁻¹. ¹HNMR (CDCl₃): 7.36 (d, J=8.5 Hz, 2 H), 6.94 (d, J=8.5 Hz, 2 H), 5.58 (D,J=15 Hz, 1 H), 5.14 (d, J=15 Hz, 1 H), 3.82 (s, 3 H), 1.97 (s, 3 H). ¹³CNMR (CDCl₃): 160.08, 143.43, 139.10, 130.20, 120.72, 114.44, 84.68,55.34, 11.04. Anal. Calcd for C₁₁H₁₂O₃S: C, 58.91; H, 5.39. Found: C,59.71; H, 5.54. EI HRMS (m/e) calcd for M+(C₁₁H₁₂O₃S) 224.0507, found224.0516.

From this, a 14% yield of the sulfenate ester4-methyl-5-(4-methoxy)phenyl-1,2-oxathiol-4-en-1-yl oxide (121) isproduced. Alternatively, treatment of the iron complex 118 with 1.5 and3 equivalents of the standard methyl cuprate generated from copper (I)iodide and methyl lithium produced sulfenate ester 121 in 23% and 22%yields respectively.

Another method of producing a desired GST-inducing sulfenate esterinvolves treating an iron sulfenate ester, such as compounds 119 and117, with 3 equivalents of the same methyl cuprate. From this reaction,the sulfenate esters4-methyl-5-(4-tert-butylphenyl-1,2-oxathiol-4-en-1-yl oxide (122) and4-methyl-5-cyclohexenyl-1,2-oxathiol-4-en-1-yl oxide (123) were bothisolated in 22% yield.

From these sequential chemical processes, the sulfenate esters producedare surprisingly effective GST, NQ0l and Ferritin H inducers. Inparticular, the sulfenate esters4-methyl-5-(4-methoxy)phenyl-1,2-oxathiol-4-en-1-yl oxide and4-methyl-5-cyclohexenyl-1,2-oxathiol-4-en-1-yl oxide exhibit high levelsof inducing activity.

4 -Methyl-5-(4-tert-butyl)phenyl-1,2-oxathiol-4-en-1-yl Oxide (122).

Iron complex 119 (0. 100 g, 0.243 mmol) was reacted with the cuprateformed from CuI (0. 139 g, 0.728 mmol) and methyllithium (1.4 M in Et₂O,1.04 ml, 1.46 mmol) using the above procedure to produce the crudeproduct which was purified to yield an off-white solid (0. 013 g, 0.052mmol, 22.0%): m.p. 73-74° C. IR (NaCl): 2954, 1123, 963, 694 cm⁻¹. ¹HNMR (CDCl₃): 7.43 (d, J=8.6 Hz, 2 H), 7.35 (d, J=8.3 Hz, 2 H), 5.60 (d,J=15.0 Hz, 1 H), 5.15 (d, J=15.1 Hz, 1 H), 1.99 (s, 3 H), 1.31 (s, 9 H).¹³C NMR (CDCl₃): 152.15, 143.91, 139.62, 128.55, 125.93, 125.61, 84.76,34.75, 31.19, 11.08. Anal. Calcd for C₁₄H₁₈O₂S: C, 67.17; H, 7.25.Found: C, 66.77; H, 7.25.

4-Methyl-5-cyclohexenyl-1,2-oxathiol4-en-1-yI Oxide (123).

Iron complex 117 (0.100 g, 0.278 mmol) was reacted with the cuprateformed from CuI (0. 159 g, 0.833 mmol) and methyllithium (1.4 M in Et₂O,1.19 ml, 1.67 mmol) using the above procedure to produce the crudeproduct which was purified to yield a colorless oil (0.012 g, 0.061mmol, 21.8%). IR (NaCl): 2924, 1123, 971, 694 cm⁻¹. ¹H NMR (CDCl₃): 5.96(m, 1 H), 5.46 (d, J=14.9 Hz, 1 H), 4.99 (d, J=14.9 Hz, 1 H), 2.17 (m, 4H), 1.92 (s, 3 H), 1.65 (m, 4 H). 13C NMR (CDCl₃): 146.22, 137.20,132.23, 127.01, 84.64, 28.55, 25.58, 22.39, 21.51, 11.23. Anal. Calcdfor C₁₀HO₂S: C, 60.58; H, 7.12. Found: C, 60.84; H, 7.27.

EXAMPLE 2 Compounds Demonstrating Metabolic Gene Induction

The compounds have been prepared and structurally characterized asdescribed in Example 1. BNL.CL2 cells were treated with Oltipraz,compound 121 described above, and compound 123 described above. Allcompounds were applied to the cells at 100 micromolar concentration for24 hours. Oltipraz induced a 340% increase in GST alpha mRNA levels.Compound 121 induced a 300% increase and compound 123 induced a 430%increase in GST alpha mRNA levels. Most of the induction occurred in thefirst 24 hours after treatment with these novel compounds but asubsequent time course showed slight additional increases in GST levels48 and 72h post treatment (See FIG. 1, for data from induction bycompound 123). Western blot analysis of the treated cells confirmed thatGST alpha (enzyme capable of performing a known GST conjugatingreaction) levels were also up in these cells (See FIG. 2). In additionto GST induction, various compounds have also been tested for theirability to induce DT-diaphorase (NQO1), and Ferritin H. The results areshown in Table 2.

The tested compounds and their chemical structures are:

TABLE 2 mRNA Induction Com- pound GST α induction NQO1 inductionFerritin H induction None − − − Oltipraz + + + t-BHQ + + + ALH-561 − − ±ALH-562 + + + ED 13.4 + + + ED 14.6 + + + DCF 070 + + + ALH-490 + + +

Cells were exposed to compounds at a final concentration of 80 μM for 24hours. RNA was isolated and analyzed by Northern blotting for theexpression of the indicated genes. Control cells were incubated withvehicle alone (0.4% DMSO). Induction indicates levels of mRNA greaterthan seen in control cells.

The data described in FIG. 1 was generated using the followingtechniques.

Northern blot: Treatment of BNL.CL2 cells with 100 μM Oltipraz, ALH-556,and ALH-562, ALH-561, ED 13.4, ED 14.6, DCF 070, ALH-490

BNL.CL2 cells (normal mouse embryonic liver cell line) were plated at˜3×10⁵ cells/ml, grown for 16-24 hours, and treated with 0 μM and 100 μMOltipraz, ALH-556 (121), and ALH-562 (123) for 24 hours. All threecompounds were dissolved in DMSO and the final DMSO concentrationpresent in all treatment conditions was 0.2%. BNL.CL2 cells wereharvested by trypsinization and total RNA was isolated using the Trizolreagent (Gibco BRL) according to the manufacturer's procedures. 15 μg ofRNA were run on a 1.1% agarose/6.6% formaldehyde gel and transferredovernight onto nitrocellulose membranes by capillary transfer using20×SSC as transfer buffer. Subsequent to RNA transfer, thenitrocellulose membrane was baked at 80° C. for 2 hours. RNA wasanalyzed using a radiolabeled mouse ferritin H and human GST-α c-DNA. Ahuman β-actin probe was used as an internal standard. The c-DNA probeswere labeled with [α³²P]dCTP by a random prime labeling. Hybridizationof the c-DNA probes to the nitrocellulose membrane was performed at 68°C. for 60 minutes using the Quick-Hyb solution (Stratagene). Membraneswere subjected to phosphoimager analysis after hybridization.

Treatment of BNL.CI2 cells with 100 μM ALH-562 for GST activitymeasurements and western blot analysis

BNL.CL2 cells were set up at ˜1×10⁵ cell/ml, grown for 16-24 hours, andtreated with 0 μM and 100 μM ALH-562. ALH-562 was dissolved in DMSO andthe final concentration of DMSO present in all treatment conditions was0.2%. After 24, 48, and 72 hours of treatment cells were harvested byscraping into ice cold 1×PBS. Cells were pelleted at 1000 rpm for 5minutes at 4° C. The pellets were frozen at −20° C. until analysis.

Cell Preparation for GST Assays

At the time of analysis, the pellet was resuspended in 1 volume of 50 mMTris/5 mM EDTA pH 7.4 and sonicated for 10-15sec. Sample was transferredto a microfuge tube and centrifuiged 13K for 10 min at 4° C. Thecytosolic supenate was used in the assays. Proteins were determined bythe method of Lowry.

GST Assay

The method used is described in Clapper et al., 1993 and Fields, et al.,1994 and is a modification of the method of Habig et al., 1974. Briefly,1-50 ul of sample was assayed at 23° C. in a solution of 0.1M potassiumphosphate, pH 6.5 and 1 mM GSH (glutathione).

The reaction was initiated with 1 mM (final) 1-chloro 2,4 dinitrobenzene(CDNB) in 20×ethanolic solution. Change in aborbance was monitored at340 nm for 90 sec. Activity was calculated using the ΔA/min andextinction coefficient, reported in nmol/min/mg protein.

GST Western Blot Assay

Cells were prepared as above. Cytosolic protein samples were boiled inLaemmli buffer for 5 min prior to loading. Samples were electrophoresedon a 12% SDS-PAGE gel and transferred by semidry electroblotting ontonitrocellulose membrane. The blot was blocked with 5% nonfat dry milk inPBS then probed with a 1:1000 dilution of affinity purified rabbitpolyclonal anti-human GST in 5% nonfat dry milk. The primary antibodieswere produced and purified as described below. The probed blot waswashed 4 times in PBS, then incubated with a 1:3000 dilution of goatanti-rabbit HRP-conjugated IgG (Cappel/ICN, Costa Mesa, Calif.). Theblot was washed again in PBS and quantitated by chemiluminescence usingX-ray film capture.

The purified antibody was prepared by coupling 25 mg of purified humanGST (alpha, mu or pi) in 5 ml 0.1M HEPES buffer, pH 7.0, to ethanolwashed Affigel-15 (Bio-Rad Hercules, Calif.), followed by blocking ofunreacted groups with 1M ethanolamine. Rabbit antisera developed againsthGST alpha, mu or pi and containing 10 mM KPO₄, 0.4M NaCl and 10 mM EDTApH 7.4 was passed over the column followed by washing with severalcolumn volumes of the same buffer containing 1M NaCl. Purifiedpolyclonal antisera monospecific for either hGST alpha, pi or mu waseluted with 0.1M glycine, pH 2.8, 1M NaCl, neutralized and dialyzedagainst 10 mM KPO₄, pH 7.4 and 50% glycerol.

EXAMPLE 3 Compositions Demonstrating anti-HIV Activity

In earlier studies, the present inventor has investigated the reactionsand interactions of S₂O and SO₂ with a variety of transition-metalcomplexes and has developed some new routes to sulfur heterocyclesynthesis based on this transition-metal chemistry (Welker, 1992). Inthese studies of new transition-metal sulfur chemistry, the aim was tobe able to (i) generate S₂O cleanly; (ii) synthesize transition-metalS₂O complexes and investigate their reaction chemistry, and (iii)investigate 3+2 cycloaddition reactions Of S₂O and SO₂ withtransition-metal propargyl, allenyl and allyl complexes.

Synthesis of an S₂O Precursor Molecule.

All of the proposed transition-metal S₂O chemistry depended on the readyavailability of an S₂O source. Prior to this work, S₂O was typicallyproduced by pyrolysis methods which produced other sulfur oxides inaddition to S₂O (Dodson et al., 1972; Murthy et al., 1971). The presentinventor developed a simple synthesis of4,5-diphenyl-1,2-dithiin-1-oxide (12) which liberated SO₂ via a retroDiels-Alder reaction (Fulcher et al., 1993; Urove et al., 1990; Uroveand Welker, 1988). Compound 12 was chosen as a potential S₂O precursorbecause it was contemplated that the steric interaction between the twophenyls in 12 would accelerate the retro Diels-Alder reaction and theresulting diene (13) should exist almost exclusively in the s-transconformation at relatively low temperatures, thereby inhibitingDiels-Alder reactions with the S₂O once it was liberated.

3+2 Cycloaddition Reactions.

The preferred synthetic methodology has been in the area oftransition-metal-mediated 3+2 cycloaddition reactions. Transition-metalpropargyl complexes react cleanly with 12 in THF at 25° C. to producemetallothiosulfinate esters (17) (Raseta et al., 1991; Raseta et al.,1989). The rates of these cycloadditions increased as thenucleophilicity of the metal propargyls (15) increased. Surprisinglythese reactions also proceeded at 25° C. and a transition-metalnucleophile induced electrocyclic ring opening of 12 was proposed toaccount for the observed products. This mechanism is consistent withMNDO predictions of the structure of 12 (12 is predicted to have a veryweak C—S bond between the CH₂ and oxidized sulfur)(Urove et al., 1990).The crystal structure of one of these metallothiosulfinate esters(17)(L_(n)M=CpFe(CO)₂, R=Ph) was also solved to rigorously prove thepresence of the S—S═O rather than O—S═S linkage in the heterocycle(Raseta et al., 1991).

The metals were removed from complexes (18) via oxidative carboxylationto produce carboethoxy substituted five-membered-ring thiosulfinateesters (S₂O)(Raseta et al., 1991; Raseta et al., 1989; Stokes et al.,1995; Ni et al., 1992).

Recently in the metal propargyl/allenyl cyclization area, studies of 3+2cycloadditions of allenyl complexes (23, 26) with SO₂ have shown thatthese cyclizations produce regioisomers (25, 28) of the propargylcyclization products (22)(Hurley et al., 1998). The structure of thecyclization product (25a) from the simplest allene was confirmed byX-ray crystallography (Hurley et al., 1998). Complex 25b was produced asa 2.3:1 mixture of diastereomers. The major diastereomer (25b anti) hadthe anti orientation of the oxygen and methyl groups as proven by shiftreagent studies (Hurley et al., 1998). Iron allenyl complexcycloaddition chemistry has not been studied to the extent propargylchemistry has (Thomasson et al., 1971; Rosenblum and Watkins, 1990).These cyclization reactions presumably proceed via intermediates (24 &27) analogous to the one (21) reported for the propargyl cyclizations(Thomasson et al., 1971).

Oxidative carboxylation was used to produce new sulfenate esters (29)and cuprates generated from MeLi or MeMgBr were used to produce thedemetallated cyclic sulfenate ester (30).

It is contemplated that saturated five membered ring sulfur heterocyclesmay also be produced using this organometallic methodology (Hayes andWelker, 1998). However, the unsaturated sulfur heterocyclic ironcomplexes appear to be much more stable than the saturated ones so thepresent example focuses on the preparation of the unsaturatedheterocycles.

Biological Screening.

Nine five membered ring thiosulfinate esters and sulfenate esters(53a-h) screened show significant cytoprotection in the XTT assay (Table3) and two of these compounds (53a and f) have EC₅₀'s below that of thebest dithiane (8, NSC 624151) described above. Based on thesepreliminary screens, it is contemplated that an aromatic or largesubstituent at R₁ (compare entries 1 & 4, 9 & 10) and a smallsubstituent at R₂ (compare entries 1, 2, & 3; 7, 8, & 9) may be best foranti-HIV-1 activity. Sulfur heterocycles containing more than 6 atomsdon't appear to be active (entries 13 & 14). In the one case where thesulfur oxidation state was examined here (entries 11 & 12) sulfuroxidation did prove to be important for zinc ejection as noted earlierfor 6, 7, 8 & 9 (Rice et al., 1997). As observed for 8 previously (Riceet al., 1997), anti-HIV-1 activity and toxicity of 53a went down whentreated with 2 equivalents of GSH (entry 6). However unlike 8, zincejecting ability of 53a was little effected by GSH. The XTTcytoprotection assay data for 53a and 53f are almost identical and thusthis data is reported (FIG. 1) for 53f as a representative example only.

(53a-g, Welker 5-ring Compounds)(7, NSC 661126)(8, NSC 624151)(53h,Welker Large Ring Compound)

TABLE 3 Antiviral Activity and NCp7 Zn ringer reactivity of rive memberring sulfur heterocycles. NCp7 Trp37 Assay (% Decrease RFU/ XTTAnti-HIV-1 30 min) Assay 50 μM EC₅₀ IC₅₀ without 50 μM Compound X R₁ R₂(μM) (μM) GSH with GSH (1) 53a S Ph CO₂Me 6 18 13.5 (2) 53b S Ph CO₂Et a45.7 6.5 (3) 53c S Ph CO₂iPr b 17.1 15.2 (4) 53d S Me CO₂Me c 16.9 14.3(5) 53e S Me CO₂iPr d 10.4 18.4 (6) 53a + S Ph CO₂Me *17.3 16.7 GSH (7)53f O Ph Me 5.9 16.9 0 (8) 53g O Ph CO₂Et e 213 26 (9) 53h O Ph CO₂Me f22.5 0 (10) 53g O Me CO₂Me g 5.3 0.2 NSC661126 9.8 30 18 (7) NSC6241516.6 184 17 (8) NSC624151 64.2 >200 /GSH

^(a) Maximum cytoprotection 31.6% @ 10 μM. ^(b) Maximum 43.3%cytoprotection @ 10 μM.

^(c) Maximum 41.4%

cytoprotection @ 10 μM. ^(d) Maximum 40% cytoprotection @ 10 μM. ^(e)Maximum 12%

cytoprotection @ 100 μM. ^(f) Maximum cytoprotection 29% @ 10 μM. ^(g)Maximum 32.6%

cytoprotection @ 3.2 μM. ^(h) Maximum 31% cytoprotection @ 31.6 μM. *Maximum 44%

cytoprotection at 95 μM.

Thus, the disclosed compounds, prepared by a novel method, and includingthe newly disclosed compound 53a, as well as compound 53f, for example,are demonstrated to possess anti-HIV-1 activity.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Armstrong, Chem. Res. Toxicol., 1991, 4, 131.

Baudouy et al., Tetrahedron, 1980, 36, 189.

Bondarenko, et al., Russian Chem. Rev., 1996, 64, 147.

Brandsma and Verkruijsse, Synthesis of Acetylenes, Allenes, andCumulenes; Elsevier Publishing Co., New York, 1981, p. 65.

Bransdma, Preparative Acetylenic Chemistry; Elsevier Publishing Co.: NewYork, 1971, 267.

Braverman and Duar, J. Am. Chem. Soc., 1983, 105, 1061.

Bunting, K. D, Lindahl, R. and Townsend, A J. (1994)Oxazaphosphorine-specific resistance in human MCF-7 breast carcinomacell lines expressing transfected rat class 3 aldehyde dehydrogenase. J.Biol Chem 269 (37), pp. 23197-23206.

Clapper, M. L, Everley, L. C. Strobel, L. A, Townsend, A. J. andEngstrom P. F. (1993) Coordinate induction of glutathione S-transferaseα, μ, and π expression in murine liver after a single administration ofoltipraz. Mol Pharm 45, pp. 469-474.

Dimitrov et al., “clinical Pharmacology Studies of Oltipraz—A PotentialChemopreventive Agent,” Invest. New Drugs, 10:289-98, 1992

Dieter, et al., Tetrahedron Lett., 1997, 38, 783.

Emester, L. (1967) DT diaphorase. Meth Enzymol 10 pp. 309-317.

Ernester, L. (1987) DT diaphorase, a historical review. Chem Scripta 27App. 1-13.

Fields, W. R., Li, Ying, and Townsend, A J. Protection by TransfectedGlutathione S-Transferase Isoenzymes Against Carcinogen-InducedAlkylation of Cellular Macromolecules in Human MCF-7 Cells.Carcinogenesis, 15: 1155-1160, 1994.

Foxman et al., J. Am. Chem. Soc., 1977, 99, 2160.

Habig, W., Pabst, M. and Jakoby, W. (1974) Glutathione s-transferase:the first enzymatic step in mercapturic acid formation J. Biol. Chem.249, pp. 7130-7139.

Harding, D. , Jackson, M. R., Wooster, R., Foumel-Gigleux, S., andBurchell, B. (1988) Cloning and substrate specificity of an human phenolUDP-glucuronosyltransferase expressed in COS-7 cells. Proc Natl Acad SciUSA 85 pp. 8381-8385.

Harpp et al., J. Org. Chem., 1976, 41, 3987.

Harris et al., J. Chem. Soc. Perk. Trans. I, 1976, 1612.

Jolly and Pettit, J. Organometallic Chem., 1968, 12, 491.

Jung et al., J. Am. Chem. Soc., 1973, 95, 3420.

Kato and Numata, Tetrahedron Lett., 1972, 203.

Kensler et al., Chem. Res. Toxicol. 1999, 12, 113.

Kensler et al., In Food Phytochemicals for Cancer Research, Huang etal., eds.: American Chemical Soc. 1994, 154.

Kessler, F. K. and Ritter J. K. (1997) Induction of a rat liverbenzo[a]pyrene-trans-7,8-dihydrodiol glucuronidating activity byoltipraz and β-naphthoflavone. Carcinogenesis 18 (1) pp. 107-114.

King, et al., Can. J. Chem., 1970, 48, 3704.

Kondo et al., J. Org. Chem., 1997, 62, 6507

Legler et al.,Tetrahedron Lett., 1972, 3907.

Liskamp et al., J. Org. Chem., 1981, 46, 5408.

Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951)Protein measurement with the Folin agents. J. Biol Chem 193 pp. 265-275.

Mahoney et al., J. Am. Chem. Soc., 1988, 110, 291

Marszak-Fleury, Ann. Chim. Paris 1958, Ser. 13, 3, 657.

Miyaura, et al., Chemistry Letters, 1979, 535.

Nemoto, N. and Gelbion, H. V. (1976) Enzymatic conjugation ofbenzo[a]pyrene oxides, phenols and dihydrodiols with UDP-glucuronicacid. Biocem Pharm 25 pp. 1221-1226.

Olah and Kuhn, J. Am. Chem. Soc., 1962, 84, 3684.

Owens, I. S. (1977) Genetic regulation of UDP-glucuronosyltransferaseinduction by polycyclic aromatic compounds in mice. J. Biol Chem. 252(9) pp. 2827-2833.

Piper et al., J. Inorg. & Nuc. Chem., 1955, 1, 165.

Ritter, J. K., Crawford, J. M., and Owens, I. S. (1991) Cloning of twohuman liver bilirubin UDPglucuronosyltransferase cDNAs with expressionin COS-1 cells. J. Biol Chem 266 pp. 1043-1047.

Roustan and Cadiot, Acad. Sci., 1969, 268, 734.

Ryter, S., Kvam, E., Richman, L, Hartmann, F., and Tyrrell, R. M. (1998)A chromatographic assay for heme oxygenase activity in cultured humancells: Application to artificial heme oxygenase overexpression.

Singh and Lippman, “Cancer Chemoprevention Part I: Retinoids andCarotenoids and other classic antioxidants,” Oncology, 12:1643-58, 1998.

Squires et al., J. Org. Chem., 46, 2373, 1981.

Summers, Protein, Sci., 1, 563, 1992.

Talalay, et al., Proc. Natl. Acad. Sci. USA, 85, 8261, 1988.

Talalay, “Mechanism of induction of enzymes that protect againstcarcinogenesis,” Adv. Enz. Reg. 28:237-50, 1989

Thoumazeau et al, Heterocycles, 19, 2247, 1982.

Vienneau, et al. “Potential genoprotective role forUDPglucuronosyltransferases in chemical carcinogenesis: Initiation ofmicronuclei by benzo(a)pyrene in UDP-glucuronosyltransferase-deficientcultured rat skin fibroblasts,” Cancer Res 55:1045-51, 1995.

Vreman and Stevenson “Heme oxygenase activity as measured by carbonmonoxide production,” Anal. Biochem 168:31-38, 1988.

Wadsworth et al., J. Org. Chem., 52, 3662, 1987.

Wattenberg, “An overview of chemoprevention: current status and futureprospects,” Proc. Soc. Exp. Biol. Med., 216:133-41, 1997.

Wawerski and Basolo, Inorg. Chem. Acta, 3, 113, 1969.

Weislow et al., J. Nat. Cancer Inst., 81, 577, 1989.

Whelan, et al. “Differential increased glutathione S-transferaseactivities in a range of multidrug-resistant human cell lines,” CancerCommunications 1:359-65, 1989

Yanagawa et al., Tetrahedron Lett., 1973, 1073

Yang and Burton, Tetrahedron, 1990, 31, 1369.

What is claimed is:
 1. A composition comprising a five membered ringthiosulfinate ester, or a five-membered ring sulfenate ester, whereinthe five membered ring has structure (1)

wherein, X is O or S; R₁ is an alkyl with from 1 to 6 carbon atoms,pheryl, substituted phenyl, cyclohexenyl, or substituted cyclohexenyl,R₂ is alkyl, phenyl or CO₂R₃, wherein R₃ is alkyl or phenyl; and when Xis O and R₁ is phenyl, R₂ is not alkyl or phenyl and R₃ is not alkyl;when X is O and R₁ is methyl, R₂ is not phenyl and R₃ is not methyl; andwhen X is S and R₂ is CO₂Et, R₁ is not methyl, phenyl or isoprenyl. 2.The composition of claim 1, wherein when R₁ is a substituted phenyl,said substituted phenyl is substituted at the 4 position with a methoxy,t-butyl, alkyl ketone, sulfonamide, or trifluoromethyl group.
 3. Thecomposition of claim 1, wherein when R₁ is a substituted cyclohexenyl,said cyclohexenyl is substituted in the 2 position with a halogen, analkyl, or a phenyl.
 4. The composition of claim 1, wherein X is O, R₁ is(4-MeO)phenyl, (4-t-Bu)phenyl or cyclohexenyl and R₂ is methyl.
 5. Thecomposition of claim 4, wherein R₁ is (4-MeO)phenyl.
 6. The compositionof claim 4, wherein R₁ is (4-t-Bu)phenyl.
 7. The composition of claim 4,wherein R₁ is cyclohexenyl.
 8. The composition of claim 1, wherein X isO, R₁ is cyclohexenyl, and R₂ is alkyl or phenyl.
 9. The composition ofclaim 8, wherein R₂ is methyl.
 10. The composition of claim 1, wherein Xis O, R₁ is (4-MeO)phenyl and R₂ is CO₂R₃.
 11. The composition of claim1, wherein X is O, and R₁ is (4-trifluoromethyl)phenyl.
 12. Thecomposition of claim 1, wherein X is O, and R₁ is phenyl (4-alkylketone).
 13. The composition of claim 1, wherein X is O, and R₁ isphenyl (4- sulfonamide).
 14. The composition of claim 1, wherein X is O,and R₁ is a cyclohexenyl with a halogen substitution at the 2 position.15. The composition of claim 14, wherein said halogen is chloride,fluoride, bromide or iodide.
 16. The composition of claim 1, wherein Xis O, R₁ is a cyclohexenyl with an alkyl substitution at the 2 position.17. The composition of claim 16, wherein said alkyl is an alkylcomprising 1 to 6 carbon atoms.
 18. The composition of claim 1, whereinX is O, and R₁ is a cyclohexenyl with a phenyl substitution at the 2position.
 19. The composition of claim 1, wherein X is S, R₁ is phenylor methyl, R₂ is CO₂R₃ and R₃ is phenyl.
 20. The composition of claim19, wherein R₁ is phenyl.
 21. The composition of claim 19, wherein R₁ ismethyl.
 22. The composition of claim 1, wherein X is S, R₁ is(4-MeO)phenyl, (4-t-Bu)phenyl or cyclohexenyl and R₂ is methyl.
 23. Thecomposition of claim 22, wherein R₁ is (4-MeO)phenyl.
 24. Thecomposition of claim 22, wherein R₁ is (4-t-Bu)phenyl.
 25. Thecomposition of claim 22, wherein R₁ is cyclohexenyl.
 26. The compositionof claim 1, wherein X is S, R₁ is phenyl, and R₂ is alkyl.
 27. Thecomposition of claim 26, wherein R₂ is methyl, ethyl, propyl, orisopropyl.
 28. The composition of claim 1, wherein X is S, R₁ iscyclohexenyl, and R₂ is alkyl or phenyl.
 29. The composition of claim28, wherein R₂ is methyl.
 30. The composition of claim 1, wherein X isS, R₁ is (4-MeO)phenyl and R₂ is CO₂R₃.
 31. The composition of claim 1,wherein X is S, and R₁ is (4-trifluoromethyl)phenyl.
 32. The compositionof claim 1, wherein X is S, and R₁ is phenyl (4-alkyl ketone).
 33. Thecomposition of claim 1, wherein X is S, and R₁ is phenyl(4-sulfonamide).
 34. The composition of claim 1, wherein X is S, and R₁is a cyclohexenyl with a halogen substitution at the 2 position.
 35. Thecomposition of claim 34, wherein said halogen is chloride, fluoride,bromide or iodide.
 36. The composition of claim 1, wherein X is S, R₁ isa cyclohexenyl with an alkyl substitution at the 2 position.
 37. Thecomposition of claim 36, wherein said alkyl is an alkyl comprising 1 to6 carbon atoms.
 38. The composition of claim 1, wherein X is S, and R₁is a cyclohexenyl with a phenyl substitution at the 2 position.
 39. Acomposition of claim 1, wherein the ring comprises an additional elementY as shown in structure (II):


40. The composition of claim 39, wherein Y is O.
 41. The composition ofclaim 40, wherein R₁ is phenyl and R₂ is methyl or phenyl.
 42. Apreparation comprising a composition of claim 1 contained in apharmaceutically acceptable carrier.
 43. A preparation of claim 42,wherein said preparation is formulated for oral administration.
 44. Apharmaceutical composition effective to inhibit an incidence of cancerin a subject when administered to said subject, said compositioncomprising a five membered ring thiosulfinate ester, or a five-memberedring sulfenate ester, wherein the five membered ring has structure (II);

and further wherein when X is O or S; R₁ is methyl, phenyl, substitutedphenyl, cyclohexenyl, or substituted cyclohexenyl; R₂ is methyl, phenyl,hydrogen or CO₂R₃, wherein R₃ is alkyl or phenyl; and Y is not presentor Y is O.
 45. The pharmaceutical composition of claim 44, wherein saidpharmaceutical composition is further defined as comprising a unit doseeffective to induce expression of a Phase II response enzyme in saidsubject when said composition is administered to said subject on aonetime or periodic basis.
 46. The pharmaceutical composition of claim45 wherein said Phase II response enzyme is glutathione-S-transferase(GST), DT-diaphorase (NQO1), or Ferritin H.
 47. The pharmaceuticalcomposition of claim 44, wherein said subject is an unaffected (normal)individual.
 48. The pharmaceutical composition of claim 44, wherein saidsubject has been exposed to, or is susceptible to being exposed to acarcinogenic xenobiotic agent.
 49. The pharmaceutical composition ofclaim 44, wherein said subject is a human subject.
 50. Thepharmaceutical composition of claim 44, wherein said pharmaceuticalcomposition is formulated to be administered in an amount of from about1 to about 1000 mg per unit dose.
 51. The pharmaceutical composition ofclaim 44, wherein said pharmaceutical composition is formulated to beadministered in an amount of from about 5 to about 600 mg per unit dose.52. The pharmaceutical composition of claim 44, wherein saidpharmaceutical composition is formulated to be administered in an amountof from about 100 to about 500 mg per unit dose.
 53. A pharmaceuticalcomposition effective to inhibit HIV replication in a subject when saidcomposition is administered to said subject, said composition comprisinga five membered ring thiosulfinate ester, or a five-membered ringsulfenate ester, wherein the five membered ring has structure (II);

and further wherein when. X is O or S; R₁ is methyl, phenyl, substitutedphenyl, cyclohexenyl, or substituted cyclohexenyl; R₂ is methyl, phenyl,hydrogen or CO₂R₃, wherein R₃ is alkyl or phenyl; and Y is not presentor Y is O.
 54. The pharmaceutical composition of claim 53, fisherdefined as a pharmaceutical package comprising a unit dose effective toinhibit HIV replication when said composition is administered to asubject periodically.
 55. The pharmaceutical composition of claim 53,wherein said subject is a human HIV patient.
 56. The pharmaceuticalcomposition of claim 53, wherein said pharmaceutical composition isformulated to be administered in an amount of from about 1 to about 1000mg per unit dose.
 57. The pharmaceutical composition of claim 53,wherein said pharmaceutical composition is formulated to be administeredin an amount of from about 5 to about 600 mg per unit dose.
 58. Thepharmaceutical composition of claim 53, wherein said pharmaceuticalcomposition is formulated to be administered in an amount of from about100 to about 500 mg per unit dose.